Grafts consisting of entire organs, composite tissues or a collection of cells are often preserved via cold storage, cryopreservation or vitrification before they are transplanted or transferred in animals including humans. Such grafts include entire heart, uterus or kidney (most often kept under hypothermic conditions, e.g., a temperature of 4° C.), composite tissues like fibrocartilage tissue (e.g., meniscus), bone, blood vessels, skin, cornea, nervous tissues, ovarian tissues or embryos (mostly cryopreserved at −80° C., −196° C., or intermediate temperatures), and hematopoietic cells, hepatocytes (liver cells) and oocytes (mostly cryopreserved, some also preserved by the process of vitrification, a procedure that avoids ice crystal formation).
While cooling in general is necessary to preserve some degree of cell viability within the graft, as a result of slowing down all metabolic, signaling and potentially toxic chemical processes, the procedure is intrinsically associated with various degrees of cell stress that may ultimately result in cell damage and cell death. Some chemicals, called “antifreeze” or “cryoprotectants,” are commonly used to lower the melting point of water and as such diminish the damage due to freezing. The main goal of adding cryoprotectants is to prevent intracellular ice crystal formation; in most cases, extracellular ice formation is more or less acceptable but intracellular ice formation is to be avoided at all cost. The reason is that ice crystals expel solute and thereby provoke hyperosmolarity in the remaining water solution, leading to water shifts between the intra- and extracellular compartment. Both crystal formation and osmolarity changes are important stress conditions for the cells that negatively influence their viability potential.
Vitrification tries to prevent crystal formation by an extremely fast temperature lowering, thereby limiting water flow and, hence, its availability to be incorporated into crystals. This procedure is ideal for cells, typically for oocytes, but not for organs or composite tissues. In any case, during thawing, re-crystallization can occur (also after vitrification) and this may add additional cell stress. Furthermore, some of the cryoprotectants, e.g., dimethylsulfoxide (DMSO), have intrinsic toxic effects on the graft cells and are known to have adverse effects on specific biological targets.1-3 Therefore, measures need to be taken that counteract or prevent some of the negative consequences of the cell-stressor conditions associated with cryopreservation or vitrification.
Additionally, hypothermic preservation is also associated with cell stress that relates in the very first place to the presence of an ischemic condition, called cold ischemia. Indeed, hypothermic preservation is mostly used for organs, and because there is no blood flow during preservation, the organ is manifestly in ischemia (the consequence of a compromised/absent perfusion). Ischemia is of particular interest here because it is one of the strong stimuli leading to the opening of connexin and pannexin hemichannels and to the propagation of cell death via gap junction channels (see further). Ischemia is a problem for organs but is less of a problem for thin composite tissues or cells.
In the two latter cases, hypothermia is associated with another stressor: the cooling (without freezing) results in a stronger inhibition of active processes, i.e., high Q10 value ATP-consuming processes, as compared to diffusive processes characterized by a Q10 close to 1. Thus, major diffusive pathways will still operate while active processes necessary to maintain homeostasis are inhibited. This will result in impaired homeostasis and cell stress. Connexin and pannexin channels (see further) constitute a highly conductant diffusive pathway. Gap junction channels composed of connexins connect the cytoplasm of neighboring cells while hemichannels composed of connexins or pannexins form membrane channels that function as a toxic pore when open. In hypothermia, as well as in cryopreservation and vitrification, ionic and molecular fluxes through these channels may considerably contribute to cell damage. Thus, hypothermia, cryopreservation and vitrification are all associated with particular stressor conditions that inevitably lead to cell dysfunction and cell death.
The case of blood vessel grafts, called vascular grafts, is particularly interesting because these grafts have wide surgical applications, but at the same token, their preservation has faced some of the prototypical problems of cryopreserving composite tissues. These grafts may be preserved by hypothermia, but in most cases, cryopreservation is applied; unfortunately, the latter procedure is still unsatisfactory because of the post-grafting complications of thrombosis and vasospasms that may occur, leading to late graft failure.4 The risk for thrombosis is enhanced by rejection, leading to loss of endothelial lining and function, and in a later phase, by immune-related intimal hyperplasia and fibrosis.5 Even with the introduction of immunosuppressive treatment,6 the improvement of harvesting techniques and preservation fluids,7 and the use of anticoagulation therapy,8 the patency of these allografts was not improved in most studies.
The performance of cryopreserved vascular grafts is suboptimal, mainly because of spontaneous vessel wall fractures9 appearing at the time of thawing or grafting. Another important issue is that the current methods of vascular cryopreservation used in most vascular banks lead to a certain degree of loss of the intimal endothelial layer.10 Before arterial or venous grafts are implanted to bypass occluded coronary arteries, they undergo extensive apoptotic and necrotic cell death in the intimal and medial layers.11 Cell death plays an important role in vascular graft failure, and important for the present study, cell-cell communication may act to expand the cell death process to neighboring healthy cells, leading to bystander cell death during cryopreservation and thawing.12 
The most direct form of cell-cell communication is provided by gap junctions that consist of intercellular channels composed of connexin proteins, named according to their predicted molecular weight.13 Vascular cells abundantly express connexins, with Cx37, Cx40 and Cx43 as the most representative isoforms.14 Gap junction channels are formed by the interaction of two hemichannels belonging to the membranes of adjacent cells and directly connect the cytoplasm of neighboring cells. Gap junctions are high-conductance channels that are able to pass cell death messengers leading to bystander cell death.12 Recently, it has been demonstrated that the physiological messenger inositol 1,4,5 trisphosphate (IP3), which can pass through gap junction channels, becomes a crucial cell death-communicating messenger under pro-apoptotic conditions.15 Additionally, hemichannels, which are half gap junction channels, may by themselves promote cell death. Unapposed hemichannels in the plasma membrane are typically closed and only open when they become incorporated into a gap junction. However, unapposed hemichannels not assembled into gap junctions may open under certain conditions like cell depolarization, decreased extracellular calcium, changes in intracellular calcium concentration, alterations in phosphorylation or redox status, mechanical strain, and ischemic or inflammatory conditions.16-20 Hemichannels are non-selective channels that allow, like gap junctions, the passage of up to 1.5 kDa molecules. As a consequence, uncontrolled hemichannel opening may lead to cell death as a toxic pore, caused by excessive diffusive fluxes, most notably the entry of sodium and calcium ions and the loss of cellular ATP or other crucial metabolic molecules.21-23 Hemichannels can also be composed of pannexins, another class of channel-forming proteins characterized by a membrane topology similar to connexins (but without sequence homology), that, when open, can accelerate or trigger cell death.24-27 
Gap junction channels and connexin hemichannels can be inhibited by connexin mimetic peptides such as Gap26 and Gap27.28-30 These peptides are identical to sequences of certain well-defined domains on the extracellular loops of the connexin protein; they first inhibit unapposed hemichannels that have their extracellular loops freely available for peptide interactions, followed by a somewhat delayed inhibition of gap junctions.22, 31, 32 Previous work has demonstrated that connexin mimetic peptides prevent the propagation of cell death by inhibiting both gap junctions and (unapposed) hemichannels.22 Recent work has furthermore shown that these peptides can significantly improve the outcome after experimental ischemia applied to heart or brain.33, 34 In the context of tissue preservation, Xu et al.35 have recently demonstrated that overexpression of Cx43 can protect cardiomyocytes against cold storage. However, the latter observation is opposite to this disclosure that relates to the block of gap junctions and hemichannels as a way to protect the cells against cell death and cell dysfunction caused by hypothermia or cryopreservation/vitrification and the associated phase of thawing.
Non-peptide inhibitors of connexin channels also exist: these include antibodies directed against the connexin protein and a large class of chemically diverse compounds that non-selectively inhibit connexin channels. Antibodies, including nanobodies, against the connexin protein display strong selectivity toward certain connexin subtypes; by contrast, several compounds exist that have multiple targets in addition to connexin channels. These include glycyrrhetinic acid and its derivative carbenoxolone, long-chain alcohols like heptanol and octanol, halogenated general volatile anesthetics like halothane, fatty acids like arachidonic acid and oleic acid, fatty acid amides like oleamide and anandamide, fenamates (arylaminobenzoates) like flufenamic acid, niflumic acid and meclofenamic acid, 5-Nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB), disodium 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (DIDS), quinine, quinidine and quinine-derivatives like mefloquine, 2-aminoethoxydiphenyl borate (2-APB), polyamines like spermine and spermidine, and certain triphenylmethanes, triphenylethanes, triarylmethanes and cyclodextrins (reviewed in ref.36). Pannexin hemichannels are blocked by some of the non-specific connexin channel blockers such as carbenoxolone, NPPB and DIDS, by disodium 4-acetamido-4′-isothiocyanato-stilben-2,2′-disulfonate (SITS), indanyloxyacetic acid (IAA-94), probenecid and by 10Panx1 peptide.37 
Considering all evidence available, there is an urgent need to improve preservation methods of grafts prior to implanting them into patients or animals.